RUNX1 and CBFB are not only important for leukemogenesis but they are also key regulators of normal hematopoiesis. These two genes are required during the earliest steps of hematopoietic stem cell formation and in subsequent stages of several blood lineages. Multiple studies suggest that dysregulation of the normal transcriptional program controlled by RUNX1 and CBFB is likely to be an important mechanism for leukemogenesis. Therefore, better understanding of the RUNX1/CBFB transcriptional program and the roles of RUNX1 and CBFB in normal hematopoiesis will lead to better understanding of the mechanisms for leukemogenesis. We have been pursuing two specific aims in this project in the last fiscal year. In the first specific aim, we have been studying the role of RUNX1 in the formation of hematopoietic stem cells (HSCs) in zebrafish. In particular, we tried to discover how HSCs could form in the absence of RUNX1. Previous studies suggest that RUNX1 is required for the emergence of definitive hematopoietic stem cells (HSCs) from the hemogenic endothelium during embryo development. For example, Runx1 knockout mouse embryos lack all definitive blood lineages and cannot survive past embryonic day 13. Surprisingly, we previously discovered that zebrafish homozygous for an ENU-induced nonsense mutation in runx1 (runx1W84X/W84X) were able to recover from a larval bloodless phase and develop to fertile adults with multi-lineage hematopoiesis, suggesting the formation of runx1-independent adult HSCs. However, our finding was based on a single zebrafish mutant line, which requires verification in independent mutants. In order to further investigate if a RUNX1-independent pathway exists for the formation of adult HSCs, we have generated three new runx1 mutants using the TALEN and CRISPR-Cas9 technologies. Two of the runx1 mutant lines carry mutations in exon 4 (a deletion of 8 bp, runx1del8/del8, and a deletion of 25 bp, runx1del25/del25), both of which truncate the runt-homologous domain. The third mutation is a large deletion of exons 3 through 8 (runx1del(e3-8)/del(e3-8)), which removes most of the coding region of runx1. All three runx1 mutant lines recapitulated our previous observations as the runx1 mutant embryos lacked the expression of the HSC marker c-myb and failed to initiate definitive hematopoiesis during early embryonic development. However, approximately 40% of the runx1-/- embryos developed into fertile adults with circulating blood cells of multi-lineages, further supporting the presence of RUNX1-independent mechanisms for the generation of HSCs. Live confocal imaging revealed the presence of hematopoietic progenitor cells in the runx1- /- mutants at early stages of embryonic development. Transcriptional profiling of these hematopoietic precursors at both bulk and single cell levels by RNAseq showed that the runx1-null hematopoietic progenitors are different from wildtype cells in global gene expression. On the other hand, the transcriptional profile of the hematopoietic cells in adult kidney is similar between runx1-/- and wildtype adult fish, except for some key myeloid and thrombocyte genes, which were downregulated in the runx1- /- mutants. Taken together, we can now provide four independent evidences that RUNX1-independent pathways for HSC formation and definitive hematopoiesis exist. In the second aim, we have been using human induced pluripotent stem cells (iPSCs) to study the function of RUNX1 in human hematopoiesis. We have been working on the culturing conditions for the differentiation of iPSCs to hematopoietic cells, in collaboration with scientists at the National Center for Advancing Translational Sciences. Hematopoietic diseases are an attractive target for iPSC-based cell therapy, because of the ability of hematopoietic stem cells (HSCs) to reconstitute the entire hematopoietic system. However, directing differentiation of iPSCs towards transplantable HSCs has proven to be difficult. In order to make it possible to treat patients suffering from blood disorders using patient-specific iPSCs, there is a great need to establish efficient methods to differentiate human iPSCs to HSCs. iPSC reporter lines that will allow testing and monitoring of directed hematopoietic differentiation of iPSCs to an HSC fate can be very useful for developing such methods. We hypothesize that RUNX1 is a good marker gene for HSC formation since RUNX1 is among the first expressed genes when hemogenic endothelial cells become committed to HSCs. Therefore, we have developed human RUNX1 reporter iPSC lines in which either the luciferase or the tdTomato cassette is inserted to the RUNX1 locus through genome editing using Zinc Finger Nucleases. These RUNX1-reporter lines have been shown to express RUNX1-luciferase or RUNX1-tdTomato protein upon hematopoietic differentiation. We have performed small compound library screening using LOPAC1280 and NCATS Pharmaceutical Collection (NPC) with the established RUNX1-reporter iPSC lines to identify compounds that enhance hematopoietic differentiation. We identified and validated 120 small compounds that were able to enhance RUNX1 expression from the RUNX1-reporter iPSC lines. The top compounds were then further tested for their potential to stimulate hematopoietic differentiation in iPSCs and in zebrafish. Several compounds were able to enhance hematopoietic differentiation potential, as evidenced by increased production of CD34+ cells from iPSCs in culture and increased production of cmyb+ HSCs in the AGM region in zebrafish embryos. Our results demonstrate successful establishment of RUNX1-reporter iPSC lines that can be used to optimize conditions for hematopoietic differentiation and to perform high-throughput chemical screening to identify compounds for more efficient generation of HSCs from iPSCs. Finally, we have been using genomic technology to determine the genomic integrity of iPSCs. Specifically we have been trying to address two important questions. First, if the iPSCs harbor more mutations than other cultured cells due to reprogramming process; and second, where the mutations coming from. From the same fibroblast populations we generated iPSCs and fibroblast subclones, which are identical to each other in terms of their tissue origin and the way they were derived, except for the treatment of reprogramming factors in the case of the iPSC generation. We then performed NextGen sequencing analysis of the iPSCs and fibroblast subclones to detect mutations. Using this approach we were able to compare the mutation profile of the iPSCs with that of the fibroblast subclones, and provide a definitive answer tothe question of if iPSCs have increased mutation burden. Our data reveal that iPSCs have comparable numbers of mutations as their sister fibroblast subclones. Moreover, we demonstrated that >90% of the mutations detected in the iPSCs and the fibroblast subclones were rare, pre-existing, mosaic variants in the parental fibroblast population. Our data therefore strongly demonstrate that iPSC reprogramming is not mutagenic and iPSCs do not contain increased mutation burden. A manuscript reporting these findings has been published earlier this year (Kwon et al., PNAS 114:1964, 2017).